This renewal proposal aims to tissue engineer an anisotropic meniscus construct that also captures the regional variations present in the native tissue. Subsequently, the construct will be implanted in a leporine model, using both allogenic and xenogenic cell sources, to achieve both meniscus repair and replacement. It is hypothesized that: 1) regionally variant, anisotropic, meniscus-shaped constructs can be engineered by optimizing cell culture and scaffoldless culture conditions;2) the synergistic and strategic temporal application of certain anabolic and catabolic stimuli will enhance the functional properties of the maturing meniscus construct;and 3) both allogenic and xenogenic constructs can be successfully implanted in a leporine model. These hypotheses will be tested via the following three specific aims: 1) to create an anisotropic meniscus construct with regional variations mimicking native tissue, 2) to enhance functional and organizational properties of constructs via synergistic, temporally coordinated exogenous stimulation, and 3) to develop tissue-construct surgical fixation techniques and implant allogenic and xenogenic constructs in the rabbit. In the previous grant, the native meniscus was found to be highly anisotropic and regionally variant both morphologically and biomechanically, motivating our current tissue engineering approach to mimic these characteristics. Aim 1 will thus use co-cultures, meniscus-specific molds, and novel seeding techniques to accomplish this goal. Also identified in the previous grant were stimuli that had positive effects on a scaffold- based approach, but in parallel studies, it was clearly demonstrated that a scaffoldless approach, driven by the differential adhesion hypothesis, was superior. Aim 2 of this proposal will use such a scaffoldless approach, in conjunction with hydrostatic pressure, tension-compression, well confinement time, TGF-1, hypoxia, and chondroitinase-ABC to create tissue engineered constructs. Furthermore, the previous grant underscored the scarcity of meniscus cells that could be used in an in vivo study. Thus, in this proposal, to avoid the use of primary cells, we will investigate the use of passaged allogenic cells and, for the first time, a xenogenic cell source in the in vivo repair and replacement of the meniscus (Aim 3). By examining both allogenic and xenogenic cell sources, this proposal seeks to obviate the issue of tissue scarcity (either autologous or allogenic meniscus and cartilage) and aims to provide a solution to the complex problem of meniscus regeneration. PUBLIC HEALTH RELEVANCE: Establishing the means to tissue engineer and implant meniscus tissue would bode well for over one million Americans that undergo meniscal procedures annually. However, the highly anisotropic mechanical properties and morphological regional variance observed in native tissue render recapitulating these structure/function relationship a complex problem. The current tissue engineering approach seeks to mimic these characteristics to produce anisotropic, inhomogeneous allogenic and/or xenogenic constructs that can restore the functional properties of the knee meniscus. Successful completion of this proposal will establish a framework for future scaffoldless meniscus regeneration attempts using alternate cell sources for the repair and replacement of meniscus tissue.